G. D. A. Gastal

The Mare Model to Study the Effects of Ovarian Dynamics on Preantral Follicle Features

Ovarian tissue collected by biopsy procedures allows the performance of many studies with clinical applications in the field of female fertility preservation. The aim of the present study was to investigate the influence of reproductive phase (anestrous vs. diestrous) and ovarian structures (antral follicles and corpus luteum) on the quality, class distribution, number, and density of preantral follicles, and stromal cell density. Ovarian fragments were harvested by biopsy pick-up procedures from mares and submitted to histological analysis. The mean preantral follicle and ovarian stromal cell densities were greater in the diestrous phase and a positive correlation of stromal cell density with the number and density of preantral follicles was observed. The mean area (mm2) of ovarian structures increased in the diestrous phase and had positive correlations with number of preantral follicles, follicle density, and stromal cell density. Biopsy fragments collected from ovaries containing an active corpus luteum had a higher follicle density, stromal cell density, and proportion of normal preantral follicles. In conclusion, our results showed: (1) the diestrous phase influenced positively the preantral follicle quality, class distribution, and follicle and stromal cell densities; (2) the area of ovarian structures was positively correlated with the follicle and stromal cell densities; and (3) the presence of an active corpus luteum had a positive effect on the quality of preantral follicles, and follicle and stromal densities. Therefore, herein we demonstrate that the presence of key ovarian structures favors the harvest of ovarian fragments containing an appropriate number of healthy preantral follicles.

Preantral follicle density in ovarian biopsy fragments and effects of mare age

The aims of the present study were to: (1) evaluate preantral follicle density in ovarian biopsy fragments within and among mares; (2) assess the effects of mare age on the density and quality of preantral follicles; and (3) determine the minimum number of ovarian fragments and histological sections needed to estimate equine follicle density using a mathematical model. The ovarian biopsy pick-up method was used in three groups of mares separated according to age (5-6, 7-10 and 11-16 years). Overall, 336 preantral follicles were recorded with a mean follicle density of 3.7 follicles per cm2. Follicle density differed (P < 0.05) among animals, ovarian fragments from the same animal, histological sections and age groups. More (P < 0.05) normal follicles were observed in the 5-6 years (97%) than the 11-16 years (84%) age group. Monte Carlo simulations showed a higher probability (90%; P < 0.05) of detecting follicle density using two experimental designs with 65 histological sections and three to four ovarian fragments. In summary, equine follicle density differed among animals and within ovarian fragments from the same animal, and follicle density and morphology were negatively affected by aging. Moreover, three to four ovarian fragments with 65 histological sections were required to accurately estimate follicle density in equine ovarian biopsy fragments.

Follicle vascularity coordinates corpus luteum blood flow and progesterone production

Colour Doppler ultrasonography was used to compare the ability of preovulatory follicle (POF) blood flow and its dimensions to predict the size, blood flow and progesterone production capability of the subsequent corpus luteum (CL). Cows (n = 30) were submitted to a synchronisation protocol. Follicles ≥7 mm were measured and follicular wall blood flow evaluated every 12 h for approximately 3.5 days until ovulation. After ovulation, cows were scanned daily for 8 days and similar parameters were evaluated for the CL. Blood samples were collected and plasma progesterone concentrations quantified. All parameters were positively correlated. Correlation values ranged from 0.26 to 0.74 on data normalised to ovulation and from 0.31 to 0.74 on data normalised to maximum values. Correlations between calculated ratios of both POF and CL in data normalised to ovulation and to maximum values ranged from moderate (0.57) to strong (0.87). Significant (P < 0.0001) linear regression analyses were seen in all comparisons. In conclusion, higher correlations were observed between the dimensions of POF and/or CL and blood flow of both structures, as well as POF and/or CL blood flow with plasma progesterone concentrations of the resultant CL. These findings indicate that follicle vascularity coordinates CL blood flow and progesterone production in synchronised beef cows.

Effects of Cryoprotectant Agents on Equine Ovarian Biopsy Fragments in Preparation for Cryopreservation

&NA; The exposure effect of cryoprotectant agents (CPAs) on morphology of preantral follicles (PAFs), stromal cell and PAF densities, and area of equine ovarian fragments were evaluated. Three independent experiments with identical methodologies were performed. Each experiment was composed of one CPA (dimethyl sulfoxide, ethylene glycol, or propylene glycol) and was performed in three replicates. Ovarian biopsy fragments were harvested from six mares in each experiment and submitted to the cryoprotectants using four times of exposure (0, 10, 15, and 20 minutes). PAF and stromal cell densities, and area of the fragments were not affected (P > .05) by any of the CPAs throughout the time of exposure. However, the morphology of the PAFs was affected (P < .05) by the CPAs. In the propylene glycol and dimethyl sulfoxide, higher (P < .05) percentages of abnormal PAFs were observed at 10 and 20 minutes of exposure, respectively. The PAF morphology in the ethylene glycol treatments was not affected (P > .05) throughout the times of exposure. Positive correlations (r = 0.57–0.77; P < .001, power = 96%–99%) were identified between PAF density and stromal cell density in all experiments. In conclusion, (1) ethylene glycol seems to be a less harmful CPA to equine PAFs, (2) exposure to CPAs did not affect the cell density and area of ovarian fragments, (3) PAF density was positively correlated with stromal cell density, and (4) stromal cell density did not affect the morphology of PAFs. HighlightsEffects of different cryoprotectant agents (CPAs) in equine ovarian tissue.Morphology of preantral follicles (PAFs), stromal cell and PAF densities evaluated.Ethylene glycol was the least harmful CPA and propylene glycol the most harmful.Preantral follicle and stromal cell densities were positively correlated.CPAs and exposure times did not affect ovarian cell densities and area of fragment.

Ovarian fragment sizes affect viability and morphology of preantral follicles during storage at 4°C

The method of transportation and the conditions imposed on the ovarian tissue are pivotal aspects for the success of ovarian tissue cryopreservation (OTC). The aim of this study was to evaluate the effect of the size of the ovarian tissue (e.g. whole ovary, biopsy size and transplant size) during different times of storage (0, 6, 12 and 24 h) on the structural integrity of equine ovarian tissue transported at 4°C. Eighteen pairs of ovaries from young mares (<10 years old) were harvested in a slaughterhouse and processed to simulate the fragment sizes (biopsy and transplant size groups) or kept intact (whole ovary group) and stored at 4°C for up to 24 h in α-MEM-enriched solution. The effect of the size of the ovarian tissue was observed on the morphology of preantral follicles, stromal cell density, DNA fragmentation and mitochondrial membrane potential. The results showed that (i) biopsy size fragments had more morphologically normal preantral follicles after 24 h of storage at 4°C; (ii) mitochondrial membrane potential was the lowest during each storage time when the whole ovary was used; (iii) DNA fragmentation rate in the ovarian cells of all sizes of fragments increased as storage was prolonged and (iv) transplant size fragments had increased stromal cell density during storage at cool temperature. In conclusion, the biopsy size fragment was the best to preserve follicle morphology for long storage (24 h); however, transportation/storage should be prior determined according to the distance (time of transportation) between patient and reproduction centers/clinics.

In vivo antral follicle wall biopsy: a new research technique to study ovarian function at the cellular and molecular levels

BackgroundIn vivo studies involving molecular markers of the follicle wall associated with follicular fluid (FF) milieu are crucial for a better understanding of follicle dynamics. The inability to obtain in vivo samples of antral follicle wall (granulosa and theca cells) without jeopardizing ovarian function has restricted advancement in knowledge of folliculogenesis in several species. The purpose of this study in mares was to develop and validate a novel, minimally invasive in vivo technique for simultaneous collection of follicle wall biopsy (FWB) and FF samples, and repeated collection from the same individual, during different stages of antral follicle development. We hypothesized that the in vivo FWB technique provides samples that maintain the normal histological tissue structure of the follicle wall layers, offers sufficient material for various cellular and molecular techniques, and allows simultaneous retrieval of FF.MethodsIn Experiment 1 (ex vivo), each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from 10-, 20-, and 30-mm follicles were repeatedly and simultaneously obtained through transvaginal ultrasound-guided technique.ResultsIn Experiment 1, the thickness of granulosa, theca interna, and theca externa layers was not influenced (P > 0.05) by the harvesting techniques. In Experiment 2, the overall recovery rates of FWB and FF samples were 97 and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size. The expression of luteinizing hormone receptor (LHR) was mostly confined in the theca interna layer, with the estradiol-related receptor alpha (ERRα) in the granulosa and theca interna layers. The 30-mm follicle group had greater (P < 0.05) LHR expression in the theca interna and ERRα in the granulosa layer compared to the other groups. The overall expression of LHR and ERRα, and the intrafollicular estradiol were higher (P < 0.05 – P < 0.0001) in the 30-mm follicle group.ConclusionThe in vivo technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function, and has the potential to be translated to other species, including humans.

134 RELAXIN AND ITS RECEPTORS IN MATURE CANINE SPERMATOZOA

Relaxin and its receptors (RXPF1 and RXFP2) are found in reproductive and non-reproductive tissues of both males and females, across species. In dogs, both relaxin and receptors are found in reproductive tissues of females and play important roles during pregnancy; however, their presence in male reproductive tissues remains unclear. The goal of this study was to explore the presence of both relaxin and receptors in mature dog spermatozoa. Semen samples of 4 adult (≥4 years old) dogs were harvested from a local breeder and diluted (vol:vol) with a pre-warmed Tris extender solution (pH 6.9). Samples were centrifuged and sperm pellets were resuspended in the Tris extender for motility and velocity analyses using computer-assisted sperm analyzer. Semen with over 70% motility were retained for analyses. A subset of those samples (n=4 dogs) was subjected to total RNA extraction, followed by RT-PCR to amplify relaxin and RXFP1/2 RNA transcripts, with β-actin used as the housekeeping gene. All PCR amplicons were run on a gel electrophoresis for imaging. Another subset of samples (n=4 dogs) were used for total protein extraction, followed by immunoblotting using anti-human RXFP1 and RXFP2 antibodies. Finally, the remaining subset of samples (n=4 dogs) was fixed in 4% paraformaldehyde and submitted to a standard procedure of immunofluorescence using an anti-human relaxin, and followed by fluorescein isothiocyanate-conjugated secondary antibody. Labelled spermatozoa were immediately analysed with flow cytometry and imaged with a confocal microscope. Gene expression data were collected after 40 cycles of PCR and all samples expressed β-actin. The detection of RXFP2 was observed at a low level in all samples, whereas RXFP1 and relaxin were absent. Protein analysis with western immunoblotting showed weak signal bands of both receptors in all samples; however, the band corresponding to RXFP1 (~82 kDa) appeared stronger than that of RXFP2 (~90 kDa). Flow cytometry revealed small proportions of relaxin immunopositive spermatozoa (less than 35%) that exhibited low fluorescence intensity. Confocal microcopy imaging confirmed the weak signals that were mostly located in the sperm head. Altogether, the findings showed that relaxin and its receptors RXFP1 and RXFP2 are present in mature spermatozoa of adult dogs. More studies are needed to better characterise the roles of the relaxin system on canine sperm fertility.

Ovarian features in white-tailed deer (Odocoileus virginianus) fawns and does

The knowledge about ovarian reserve is essential to determine the reproductive potential and to improve the methods of fertility control for overpopulated species, such as white-tailed deer (Odocoileus virginianus). The goal of this study was to evaluate the effect of age on the female reproductive tract of white-tailed deer, focusing on ovarian features. Genital tracts from 8 prepubertal and 10 pubertal females were used to characterize the preantral follicle population and density, morphology, distribution of follicular classes; stromal cell density; and apoptosis in the ovary. In addition, uterus and ovary weights and dimensions were recorded; and the number and the size of antral follicles and corpus luteum in the ovary were quantified. Overall, fawns had a greater (P < 0.05) preantral follicle population, percentage of normal follicles, and preantral follicle density than does. The mean stromal cell density in ovaries of fawns and does differed among animals but not between age groups. The apoptotic signaling did not differ (P > 0.05) between the ovaries of fawns and does. However, apoptotic ovarian cells negatively (P < 0.001) affected the preantral follicle morphology and density, and conversely, a positive correlation was observed with stromal cell density. As expected, the uteri and ovaries were larger (P < 0.002) and heavier (P < 0.001) in does than in fawns. In conclusion, this study has shown, for the first time, the preantral follicle population and distribution of classes, rate of morphologically normal follicles, and density of preantral follicles and stromal cells in white- tailed deer. Therefore, the findings herein described lead to a better understanding of the white-tailed deer ovarian biology, facilitating the development of new methods of fertility control.

Novel prospects for evaluation of follicle wall blood flow using color-Doppler ultrasonography

The goal of this study was to develop an objective method for evaluation of ovarian follicle wall blood flow in cattle. Two subjective methods were used: (I) real-time ultrasound evaluations performed by one operator in the barn and (II) video clip evaluations performed by four operators in the laboratory. The following objective methods evaluated in the laboratory were used for comparison: (I) percentage of follicle wall circumference under blood flow (WUF) and (II) pixel area of color-Doppler signals. Cows (n = 21) were submitted to a synchronization protocol, follicles ≥7 mm were measured, and blood flow was evaluated every 12 h until ovulation using color-Doppler ultrasonography. No difference (P > 0.05) was observed among laboratory operators from day 2 of training onwards. Therefore, an average score of all operators was used for comparisons among different methods. Both subjective and objective methods of evaluation showed an increase (P < 0.0001) in follicle blood flow over time. Higher (P < 0.001) correlations were obtained between WUF and subjective laboratory evaluation than between WUF and pixel area or WUF and subjective barn data. Higher (P < 0.0003) correlation coefficients were observed for WUF than for the pixel area when compared with the barn (r = 0.70 vs. r = 0.42) or laboratory (r = 0.84 vs. r = 0.62) data. Subjective evaluations at the laboratory and barn produced stronger correlations with WUF (P < 0.0008) than with pixel area (P < 0.01). In conclusion, WUF is an effective and reliable method for objective evaluation of follicle wall blood flow in cows.

Spatial distribution of preantral follicles in the equine ovary

Comprehensive studies on spatial distribution of preantral follicles in the ovary are scarce. Considering that preantral follicles represent the main ovarian reserve, harvesting of these follicles is crucial for the development/use of assisted reproductive techniques. Therefore, knowledge on follicle spatial distribution can be helpful for targeting areas with richer number of preantral follicles through biopsy procedures. The aim of this study was to assess the distribution and localization of equine preantral follicles according to: (i) age, (ii) ovarian portion (lateral and intermediary) and region (dorsal and ventral), (iii) distance from the geometric center, and (iv) follicular class. Ovaries from young and old mares (n = 8) were harvested in a slaughterhouse and submitted to histological processing for further evaluation. For data analyses, a novel methodology was developed according to the geometric center of each histological section for a precise determination of preantral follicle distribution. Results indicated that (i) equine preantral follicles are clustered and located near to the ovarian geometric center, and that aging induced their dispersion through the ovarian cortex; (ii) the distance from the geometric center was shorter for developing follicles than primordial; and (iii) secondary follicles were more distant from the geometric center but closer to the ovulation fossa. In conclusion, the spatial distribution of preantral follicles was successfully determined in the equine ovary and was affected by age, region, and portion.